Human carboxylesterase 2 (hCES2A), one of the major serine hydrolases distributed in the small intestine, plays a crucial role in hydrolysis of ester-bearing drugs. Accumulating evidence has indicated that hCES2A inhibitor therapy can modulate the pharmacokinetic and toxicological profiles of some important hCES2A-substrate drugs, such as the anticancer agent CPT-11. Herein, a series of indanone-chalcone hybrids are designed and synthesized to find potent and highly selective hCES2A inhibitors. Inhibition assays demonstrated that most indanone-chalcone hybrids displayed strong to moderate hCES2A inhibition activities. Structure-hCES2A inhibition activity relationship studies showed that introduction of a hydroxyl at the C4' site and introduction of an N-alkyl group at the C6 site were beneficial for hCES2A inhibition. Particularly, B7 (an N-alkylated 1-indanone-chalcone hybrid) exhibited the most potent inhibition on hCES2A and excellent specificity (this agent could not inhibit other human esterases including hCES1A and butyrylcholinesterase). Inhibition kinetic analyses demonstrated that B7 potently inhibited hCES2A-mediated FD hydrolysis in a mixed inhibition manner, with a calculated K value of 0.068 μM. Furthermore, B7 was capable of inhibiting intracellular hCES2A in living cells and displayed good metabolic stability. Collectively, our findings show that indanone-chalcone hybrids are good choices for the development of hCES2A inhibitors, while B7 is a promising candidate for the development of novel anti-diarrhea agents to ameliorate irinotecan-induced intestinal toxicity.